Competing adaptations maintain nonadaptive variation in a wild cricket population.

How emerging adaptive variants interact is an important factor in the evolution of wild populations, but the opportunity to empirically study this interaction is rare. We recently documented the emergence of an adaptive phenotype "curly-wing" in Hawaiian populations of field crickets (Teleogryllus oceanicus). Curly-wing inhibits males' ability to sing, protecting them from eavesdropping parasitoid flies (Ormia ochracea). Surprisingly, curly-wing co-occurs with similarly protective silent "flatwing" phenotypes in multiple populations, in which neither phenotype has spread to fixation. These two phenotypes are frequently coexpressed, but since either sufficiently reduces song amplitude to evade the fly, their coexpression confers no additional fitness benefit. Numerous "off-target" phenotypic changes are known to accompany flatwing, and we find that curly-wing, too, negatively impacts male courtship ability and affects mass and survival of females under lab conditions. We show through crosses and genomic and mRNA sequencing that curly-wing expression is associated with variation on a single autosome. In parallel analyses of flatwing, our results reinforce previous findings of X-linked single-locus inheritance. By combining insights into the genetic architecture of these alternative phenotypes with simulations and field observations, we show that the co-occurrence of these two adaptations impedes either from fixing, despite extreme fitness benefits, due to fitness epistasis. This co-occurrence of similar adaptive forms in the same populations might be more common than is generally considered and could be an important force inhibiting adaptive evolution in wild populations of sexually reproducing organisms.

A novel cricket morph has diverged in song and wing morphology across island populations.

Divergence of sexual signals between populations can lead to speciation, yet opportunities to study the immediate aftermath of novel signal evolution are rare. The recent emergence and spread of a new mating song, purring, in Hawaiian populations of the Pacific field cricket (Teleogryllus oceanicus) allows us to investigate population divergence soon after the origin of a new signal. Male crickets produce songs with specialized wing structures to attract mates from afar (calling) and entice them to mate when found (courtship). However, in Hawaii, these songs also attract an eavesdropping parasitoid fly (Ormia ochracea) that kills singing males. The novel purring song, produced with heavily modified wing morphology, attracts female crickets but not the parasitoid fly, acting as a solution to this conflict between natural and sexual selection. We've recently observed increasing numbers of purring males across Hawaii. In this integrative field study, we investigated the distribution of purring and the proportion of purring males relative to other morphs in six populations on four islands and compared a suite of phenotypic traits (wing morphology, calling song and courtship song) that make up this novel signal across populations of purring males. We show that purring is found in varying proportions across five, and is locally dominant in four, Hawaiian populations. We also show that calling songs, courtship songs and wing morphology of purring males differ geographically. Our findings demonstrate the rapid pace of evolution in island populations and provide insights into the emergence and divergence of new sexual signals over time.

Spatial and Sexual Divergence of Gut Bacterial Communities in Field Cricket Teleogryllus occipitalis (Orthoptera: Gryllidae).

The insect gut is colonized by microbes that confer a myriad of beneficial services to the host, including nutritional support, immune enhancement, and even influence behavior. Insect gut microbes show dynamic changes due to the gut compartments, sex, and seasonal and geographic influences. Crickets are omnivorous hemimetabolous insects that have sex-specific roles, such as males producing chirping sounds for communication and exhibiting fighting behavior. However, limited information is available on their gut bacterial communities, hampering studies on functional compartmentalization of the gut and sex-specific roles of the gut microbiota in omnivorous insects. Here, we report a metagenomic analysis of the gut bacteriome of the field cricket Teleogryllus occipitalis using 16S rRNA V3-V4 amplicon sequencing to identify sex- and compartment-dependent influences on its diversity and function. The structure of the gut microbiota is strongly influenced by their gut compartments rather than sex. The species richness and diversity analyses revealed large difference in the bacterial communities between the gut compartments while minor differences were observed between the sexes. Analysis of relative abundance and predicted functions revealed that nitrogen- and oxygen-dependent metabolism and amino acid turnover were subjected to functional compartmentalization in the gut. Comparisons between the sexes revealed differences in the gut microbiota, reflecting efficiency in energy use, including glycolytic and carbohydrate metabolism, suggesting a possible involvement in egg production in females. This study provides insights into the gut compartment dependent and sex-specific roles of host-gut symbiont interactions in crickets and the industrial production of crickets.

Variable dosage compensation is associated with female consequences of an X-linked, male-beneficial mutation.

Recent theory has suggested that dosage compensation mediates sexual antagonism over X-linked genes. This process relies on the assumption that dosage compensation scales phenotypic effects between the sexes, which is largely untested. We evaluated this by quantifying transcriptome variation associated with a recently arisen, male-beneficial, X-linked mutation across tissues of the field cricket Teleogryllus oceanicus, and testing the relationship between the completeness of dosage compensation and female phenotypic effects at the level of gene expression. Dosage compensation in T. oceanicus was variable across tissues but usually incomplete, such that relative expression of X-linked genes was typically greater in females. Supporting the assumption that dosage compensation scales phenotypic effects between the sexes, we found tissues with incomplete dosage compensation tended to show female-skewed effects of the X-linked allele. In gonads, where expression of X-linked genes was most strongly female-biased, ovaries-limited genes were much more likely to be X-linked than were testes-limited genes, supporting the view that incomplete dosage compensation favours feminization of the X. Our results support the expectation that sex chromosome dosage compensation scales phenotypic effects of X-linked genes between sexes, substantiating a key assumption underlying the theoretical role of dosage compensation in determining the dynamics of sexual antagonism on the X.

Sex-specific associations between life-history traits and a novel reproductive polymorphism in the Pacific field cricket.

Associations between heritable polymorphisms and life-history traits, such as development time or reproductive investment, may play an underappreciated role in maintaining polymorphic systems. This is because selection acting on a particular morph could be bolstered or disrupted by correlated changes in life history or vice versa. In a Hawaiian population of the Pacific field cricket (Teleogryllus oceanicus), a novel mutation (flatwing) on the X-chromosome is responsible for a heritable polymorphism in male wing structure. We used laboratory cricket colonies fixed for male wing morph to investigate whether males and females bearing the flatwing or normal-wing (wild-type) allele differed in their life-history traits. We found that flatwing males developed faster and had heavier testes than normal-wings, whereas flatwing homozygous females developed slower and had lighter reproductive tissues than normal-wing homozygous females. Our results advance our understanding of the evolution of polymorphisms by demonstrating that the genetic change responsible for a reproductive polymorphism can also have consequences for fundamental life-history traits in both males and females.

Expression of 20-hydroxyecdysone-related genes during gonadal development of Teleogryllus emma (Orthoptera: Gryllidae).

Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.

Males adjust their manipulation of female remating in response to sperm competition risk.

To reduce the potential for sperm competition, male insects are thought to inhibit the post-mating reproductive behaviour of females through receptivity-inhibiting compounds transferred in the ejaculate. Selection is expected to favour phenotypic plasticity in male post-copulatory expenditure, with males investing strategically in response to their perceived risk of sperm competition. However, the impact that socially cued strategic allocation might have on female post-mating behaviour has rarely been assessed. Here, we varied male perception of sperm competition risk, both prior to and during mating, to determine if a male's competitive environment impacts the extent to which he manipulates female remating behaviour. We found that female Australian field crickets (Teleogryllus oceanicus) mated to males that were reared under sperm competition risk emerged from a shelter in search of male song sooner than did females mated to males reared without risk, but only when mating occurred in a risk-free environment. We also found that females reared in a silent environment where potential mates were scarce emerged from the shelter sooner than females exposed to male calls during development. Collectively, our findings suggest complex interacting effects of male and female sociosexual environments on female post-mating sexual receptivity.

Sexual selection and population divergence III: Interspecific and intraspecific variation in mating signals.

A major challenge for studying the role of sexual selection in divergence and speciation is understanding the relative influence of different sexually selected signals on those processes in both intra- and interspecific contexts. Different signals may be more or less susceptible to co-option for species identification depending on the balance of sexual and ecological selection acting upon them. To examine this, we tested three predictions to explain geographic variation in long- versus short-range sexual signals across a 3,500 + km transect of two related Australian field cricket species (Teleogryllus spp.): (a) selection for species recognition, (b) environmental adaptation and (c) stochastic divergence. We measured male calling song and male and female cuticular hydrocarbons (CHCs) in offspring derived from wild populations, reared under common garden conditions. Song clearly differentiated the species, and no hybrids were observed suggesting that hybridization is rare or absent. Spatial variation in song was not predicted by geography, genetics or climatic factors in either species. In contrast, CHC divergence was strongly associated with an environmental gradient supporting the idea that the climatic environment selects more directly upon these chemical signals. In light of recently advocated models of diversification via ecological selection on secondary sexual traits, the different environmental associations we found for song and CHCs suggest that the impact of ecological selection on population divergence, and how that influences speciation, might be different for acoustic versus chemical signals.

Ochrobactrum teleogrylli sp. nov., a pesticide-degrading bacterium isolated from the insect Teleogryllus occipitalis living in deserted cropland.

A Gram-stain-negative, non-spore-forming, motile, aerobic, rod-shaped bacteria strain, designated LCB8T, was isolated from the insect Teleogryllus occipitalis captured from a deserted cropland in Shuangliu district, Chengdu, PR China. Phylogenetic analysis on the basis of 16S rRNA gene sequence indicated that the strain represented a member of the genus Ochrobactrum, family Brucellaceae, class Alphaproteobacteria. Ochrobactrum pecoris CCUG 60088T (97.9 %) and Ochrobactrum haematophilum CCUG 38531T (98.8 %) were identified as the most closely related phylogenetic neighbours of strain LCB8T. The novel strain was able to grow at salt concentrations of 0-4.5 % (w/v), pH 5-9 and temperatures of 20-42 °C. The major quinone system was ubiquinone Q-10, the major fatty acids were C18 : 1ω7c, C16 : 0 and C18 : 0. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol and four undefined aminolipids. The major polyamines were putrescine and spermidine. Genome sequencing revealed a genome size of 4.76 Mbp and a DNA G+C content of 57.1 mol%. These phenotypic, genotypic and chemotaxonomic traits excellently supported the affiliation of LCB8T to the genus Ochrobactrum. Pairwise determined whole-genome average nucleotide identity (ANI) values indicated that strain LCB8T represents a novel species, for which we propose the name Ochrobactrum teleogrylli sp. nov. with the type strain LCB8T (=KCTC 72031T=CGMCC 1.13984T).

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.

Can behaviour impede evolution? Persistence of singing effort after morphological song loss in crickets.

Evolutionary loss of sexual signals is widespread. Examining the consequences for behaviours associated with such signals can provide insight into factors promoting or inhibiting trait loss. We tested whether a behavioural component of a sexual trait, male calling effort, has been evolutionary reduced in silent populations of Hawaiian field crickets (Teleogryllus oceanicus). Cricket song requires energetically costly wing movements, but 'flatwing' males have feminized wings that preclude song and protect against a lethal, eavesdropping parasitoid. Flatwing males express wing movement patterns associated with singing but, in contrast with normal-wing males, sustained periods of wing movement cannot confer sexual selection benefits and should be subject to strong negative selection. We developed an automated technique to quantify how long males spend expressing wing movements associated with song. We compared calling effort among populations of Hawaiian crickets with differing proportions of silent males and between male morphs. Contrary to expectation, silent populations invested as much in calling effort as non-silent populations. Additionally, flatwing and normal-wing males from the same population did not differ in calling effort. The lack of evolved behavioural adjustment following morphological change in silent Hawaiian crickets illustrates how behaviour might sometimes impede, rather than facilitate, evolution.

Natural and sexual selection on cuticular hydrocarbons: a quantitative genetic analysis.

While the reproductive benefits of sexual displays have been widely studied, we have relatively limited evidence of the fitness costs associated with most display traits. Insect cuticular hydrocarbon (CHC) profiles are sexually selected traits that also protect against desiccation. These two functions are thought to oppose each other, with investment in particular compounds believed to increase attractiveness at the expense of compounds that protect against water loss. We investigated this potential trade-off in a quantitative genetic framework using the Australian field cricket, Teleogryllus oceanicus. Several compounds were significantly genetically correlated with either attractiveness or desiccation resistance. Of these compounds, one was negatively genetically correlated with attractiveness but positively genetically correlated with desiccation resistance. Furthermore, scoring each individual's overall CHC profile for its level of attractiveness and desiccation resistance indicated a negative genetic correlation between these multivariate phenotypes. Together, our results provide evidence for a genetic trade-off between sexually and naturally selected functions of the CHC profile. We suggest that the production of an attractive CHC profile may be costly for males, but highlight the need for further work to support this finding experimentally. Genetic covariation between the CHC profile and attractiveness suggests that females can gain attractive sons through female choice.

Release from intralocus sexual conflict? Evolved loss of a male sexual trait demasculinizes female gene expression.

The loss of sexual ornaments is observed across taxa, and pleiotropic effects of such losses provide an opportunity to gain insight into underlying dynamics of sex-biased gene expression and intralocus sexual conflict (IASC). We investigated this in a Hawaiian field cricket, Teleogryllus oceanicus, in which an X-linked genotype ( flatwing) feminizes males' wings and eliminates their ability to produce sexually selected songs. We profiled adult gene expression across somatic and reproductive tissues of both sexes. Despite the feminizing effect of flatwing on male wings, we found no evidence of feminized gene expression in males. Instead, female transcriptomes were more strongly affected by flatwing than males', and exhibited demasculinized gene expression. These findings are consistent with a relaxation of IASC constraining female gene expression through loss of a male sexual ornament. In a follow-up experiment, we found reduced testes mass in flatwing males, whereas female carriers showed no reduction in egg production. By contrast, female carriers exhibited greater measures of body condition. Our results suggest sex-limited phenotypic expression offers only partial resolution to IASC, owing to pleiotropic effects of the loci involved. Benefits conferred by release from intralocus conflict could help explain widespread loss of sexual ornaments across taxa.

Life history consequences of developing in anthropogenic noise.

When environments change rapidly, adaptive phenotypic plasticity can ameliorate negative effects of environmental change on survival and reproduction. Recent evidence suggests, however, that plastic responses to human-induced environmental change are often maladaptive or insufficient to overcome novel selection pressures. Anthropogenic noise is a ubiquitous and expanding disturbance with demonstrated effects on fitness-related traits of animals like stress responses, foraging, vigilance, and pairing success. Elucidating the lifetime fitness effects of noise has been challenging because longer-lived vertebrate systems are typically studied in this context. Here, we follow noise-stressed invertebrates throughout their lives, assessing a comprehensive suite of life history traits, and ultimately, lifetime number of surviving offspring. We reared field crickets, Teleogryllus oceanicus, in masking traffic noise, traffic noise from which we removed frequencies that spectrally overlap with the crickets' mate location song and peak hearing (nonmasking), or silence. We found that exposure to masking noise delayed maturity and reduced adult lifespan; crickets exposed to masking noise spent 23% more time in juvenile stages and 13% less time as reproductive adults than those exposed to no traffic noise. Chronic lifetime exposure to noise, however, did not affect lifetime reproductive output (number of eggs or surviving offspring), perhaps because mating provided females a substantial longevity benefit. Nevertheless, these results are concerning as they highlight multiple ways in which traffic noise may reduce invertebrate fitness. We encourage researchers to consider effects of anthropogenic disturbance on growth, survival, and reproductive traits simultaneously because changes in these traits may amplify or nullify one another.

Sex differences in nutrient intake can reduce the potential for sexual conflict over fitness maximization by female and male crickets.

As females and males have different roles in reproduction, they are expected to require different nutrients for the expression of reproductive traits. However, due to their shared genome, both sexes may be constrained in the regulation of nutrient intake that maximizes sex-specific fitness. Here, we used the Geometric Framework for nutrition to examine the effect of macronutrient and micronutrient intakes on lifespan, fecundity and cuticular hydrocarbons (CHCs) that signal mate quality to prospective mates in female field crickets, Teleogryllus oceanicus. In addition, we contrasted nutritional effects on life-history traits between males and females to determine how sex differences influence nutrient regulation. We found that carbohydrate intake maximized female lifespan and protein intake influenced CHC expression, while early life fecundity (cumulative fecundity at day 21) and lifetime fecundity were dependent on both macronutrient and micronutrient intakes. Fecundity required different nutrient blends to those required to optimize sperm viability in males, generating the potential for sexual conflict over macronutrient intake. The regulation of protein (P) and carbohydrate (C) intakes by virgin and mated females initially matched that of males, but females adjusted their intake to a higher P:C ratio, 1P:2C, that maximized fecundity as they aged. This suggests that a sex-specific, age-dependent change in intake target for sexually mature females, regardless of their mating status, adjusts protein consumption in preparation for oviposition. Sex differences in the regulation of nutrient intake to optimize critical reproductive traits in female and male T. oceanicus provide an example of how sexual conflict over nutrition can shape differences in foraging between the sexes.

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance.

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the "gatekeepers" of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close-range signals are as important as long-distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.

Obligately silent males sire more offspring than singers in a rapidly evolving cricket population.

How sexual traits are gained and lost in the wild remains an important question in evolutionary biology. Pacific field crickets (Teleogryllus oceanicus) in Hawaii provide an unprecedented opportunity to investigate the factors facilitating evolutionary loss of a sexual signal in real time. Natural selection from an acoustically orienting parasitoid fly drove rapid evolution of a novel, silent male morph. While silent (flatwing) males enjoy protection from the fly, they face difficulty attracting mates. We tested how offspring production varies in association with three male attributes affected by the spread of flatwing: wing morph (flatwing or normal-wing), age (flatwings should survive longer than singers) and exposure to calling song during rearing (wild populations with many flatwings lack ambient calling song). Per mating event, flatwings sired more offspring than singers and older males were mounted more quickly by females when presented with standard courtship song. Despite prior work showing that male age and acoustic experience influence sperm characteristics associated with fertilization, age and song exposure had no influence on male offspring production per mating. This represents the first evidence that the silent male morph possesses a reproductive advantage that may help compensate for precopulatory barriers to mate attraction.

Increased socially mediated plasticity in gene expression accompanies rapid adaptive evolution.

Recent theory predicts that increased phenotypic plasticity can facilitate adaptation as traits respond to selection. When genetic adaptation alters the social environment, socially mediated plasticity could cause co-evolutionary feedback dynamics that increase adaptive potential. We tested this by asking whether neural gene expression in a recently arisen, adaptive morph of the field cricket Teleogryllus oceanicus is more responsive to the social environment than the ancestral morph. Silent males (flatwings) rapidly spread in a Hawaiian population subject to acoustically orienting parasitoids, changing the population's acoustic environment. Experimental altering crickets' acoustic environments during rearing revealed broad, plastic changes in gene expression. However, flatwing genotypes showed increased socially mediated plasticity, whereas normal-wing genotypes exhibited negligible expression plasticity. Increased plasticity in flatwing crickets suggests a coevolutionary process coupling socially flexible gene expression with the abrupt spread of flatwing. Our results support predictions that phenotypic plasticity should rapidly evolve to be more pronounced during early phases of adaptation.

Purring Crickets: The Evolution of a Novel Sexual Signal.

Opportunities to observe contemporary signal change are incredibly rare but critical for understanding how diversity is created and maintained. We discovered a population of the Pacific field cricket (Teleogryllus oceanicus) with a newly evolved song (purring), different from any known cricket. Male crickets use song to attract females from afar and to court females once near. Teleogryllus oceanicus is well known for sexual signal evolution, as exemplified by a recent signal loss. In this study, we characterized the new purring sound and investigated the role of the purr in long-distance and short-distance communication. The purring sound differed from typical ancestral calls in peak frequency, amplitude, and bandwidth. Further, the long-distance purring song facilitated mate location, though the role of courtship purring song is less clear. Our discovery of purring male crickets is an unprecedented opportunity to watch the emergence of a newly evolved sexual signal unfold in real time and has potential to illuminate the mechanisms by which evolutionary novelties arise and coevolve between the sexes.

Opposing patterns of intraspecific and interspecific differentiation in sex chromosomes and autosomes.

Linking intraspecific and interspecific divergence is an important challenge in speciation research. X chromosomes are expected to evolve faster than autosomes and disproportionately contribute to reproductive barriers, and comparing genetic variation on X and autosomal markers within and between species can elucidate evolutionary processes that shape genome variation. We performed RADseq on a 16 population transect of two closely related Australian cricket species, Teleogryllus commodus and T. oceanicus, covering allopatry and sympatry. This classic study system for sexual selection provides a rare exception to Haldane's rule, as hybrid females are sterile. We found no evidence of recent introgression, despite the fact that the species coexist in overlapping habitats in the wild and interbreed in the laboratory. Putative X-linked loci showed greater differentiation between species compared with autosomal loci. However, population differentiation within species was unexpectedly lower on X-linked markers than autosomal markers, and relative X-to-autosomal genetic diversity was inflated above neutral expectations. Populations of both species showed genomic signatures of recent population expansions, but these were not strong enough to account for the inflated X/A diversity. Instead, most of the excess polymorphism on the X could better be explained by sex-biased processes that increase the relative effective population size of the X, such as interspecific variation in the strength of sexual selection among males. Taken together, the opposing patterns of diversity and differentiation at X versus autosomal loci implicate a greater role for sex-linked genes in maintaining species boundaries in this system.